1. Field of the Invention
This invention relates to the field of immunology and cancer diagnostics and therapeutics applications. More particularly, it concerns antibodies specific to mutant oncogenes but not to the native proto-oncogene, immunochemicals made from those antibodies, and diagnostic and therapeutic methods which use those immunochemicals.
2. Description of Related Disclosures
Oncogenes are cellular genes that contribute to the neoplastic transformation of cells. See generally Weinberg, R., Scientific American, 249: 126-142 (1983). They are altered forms of normal cellular genes, and have been detected in a wide range of human tumors and tumor cell lines. Oncogenes have been detected by transfecting DNA from human tumors or tumor cell lines onto a cell line known as NIH/3T3 cells; cells that take up and express oncogenes from the transfected DNA become altered morphologically, and begin to grow as dense foci. Cells from these foci are able to form tumors when grown in mice (Lane, M. A., Sainten, A. and Cooper, G. M., Proc Nalt Acad Sci USA, 78: 5185-5189 (1981); and Shilo, B. and Weinberg, R. A., Nature, 289: 607-609 (1981)).
The p21 proteins, encoded by members of the ras proto-oncogene family, cause the neoplastic transformation of certain cells in culture when activated through somatic mutation and have been implicated in the generation of a variety of human cancers. The frequency at which ras genes become activated in human tumors may be 20% or higher. Comparison of the structure of the ras oncogenes has revealed that they differ from their non-transforming normal counterparts by point mutations that alter the amino acid sequence of the gene product, p21 protein, at either position 12 or 61 (Parada, L. F., et al., Nature, 297: 474-479 (1982); Santos, E., et al., Nature, 298: 343-347 (1982); Yuasa, Y. et al., Nature, 303: 775-779 (1982)). The most frequently observed mutation leading to the oncogenic activation of the p21 proteins results in the alteration of the amino acid residue at position 12 of the protein to any of several other residues. Marshall, C. J. et al., Cancer Surveys, 3: 183-214 (1984).
The p21 protein is known to bind specifically to GTP and to hydrolyze GTP to GDP and inorganic phosphate. Shih, T. Y. et al., Nature, 287: 686-691 (1980), McGrath, J. P. et al., Nature, 310: 644-649 (1984) and Swelt, R. W. et al., Nature, 311: 273-275 (1984). In forms of p21 protein that contain a threonine residue at position 59, the phosphate is transferred to this threonine residue by an autophosphorylation reaction. McGrath et al., supra, Gibbs, J. B. et al., P.N.A.S. USA, 81: 2674-2678 (1984) and Shih, T. Y. et al., Nature (London) 287: 686-691 (1980). The role of these activities in the transforming potential of activated p21 is not yet known. It has been suggested, however, that the portion of the p21 protein surrounding position 12 might be involved in the interaction of p21 protein with GTP. Gay, N. J. et al., Nature, 301: 262-264 (1983) and Weirenga, R. K. et al., Nature, 302: 842-844 (1983). Furth, M. et al., J. Virol., 43: 294-304 (1982) disclose that both polyclonal sera and monoclonal antibodies directed against various p21 determinants allow GTP binding. The monoclonal antibody Y13-259 does not discriminate between normal and activated p21 protein.
European Patent Publication No. 108,564 to Cline et al. discloses methods for using a probe such as an antibody for detecting the expression product of a c-onc gene, including a ras oncogene, in diagnosing for the presence of malignancy. This publication does not distinguish between the wild-type and mutant form of the polypeptide. U.S. patent application Ser. No. 369,517 filed Apr. 1, 1982 entitled "Expression of Retroviral myc Genes in Human Neoplastic Cells" to T. Papas et al. discloses use of cloned recombinant DNA probes labeled with .sup.32 P containing the myc oncogene sequences of avian myelocytomatosis virus strain (MC29) to detect human tumor cells. Various blood cells were found to produce elevated amounts of RNA homologous to the myc gene sequences of MC29.
It is known that a peptide segment can be chemically synthesized which corresponds precisely with the amino acid sequence of the region of interest of a protein being studied and the peptide may be coupled to a carrier protein and injected into a suitable host to obtain antibodies. For example, Alexander et al., Nature, 306: 697-699 (1983) describe two white blood cell proteins (Thy-1) which differ by only a single amino acid and antisera specific thereto induced by short chemically synthesized peptides which span the regions of sequence variation. Tamura, T. et al., Cell, 34: 587-596 (1983) disclose the raising of antibodies against synthetic oligopeptides corresponding to the primary structure of pp60.sup.src and using the antibodies to immunoprecipitate pp60.sup.src. Lerner et al., Proc. Natl. Acad. Sci. USA, 78: 3403-3407 (1981) disclose use of peptides as vaccines where the peptides correspond to amino acid sequences predicted from the nucleotide sequences of proteins such as the hepatitis B surface antigen. See also Walter, G. et al., P.N.A.S. USA, 77: 5197-5200 ( 1980) and Lerner, R. A., Nature, 229: 592-596 (1982). As to the p21 protein, PCT W0/84/01389 published Apr. 12, 1984 to Weinberg et al. discloses the differences between the oncogene of DNA isolated from human bladder cancer cells and its corresponding proto-oncogene. In one type of assay serological reagents are described such as an antibody specific for either p21 protein expressed from the proto-oncogene or oncogene which may be used to detect carcinogenesis. The antibodies may be raised by using a chemically synthesized peptide segment corresponding to the amino acid sequence of the region of interest of the normal or altered peptide sequence being studied.
Hand, H. et al., P.N.A.S. USA, 81: 5227-5231 (1984) disclose the generation of monoclonal antibodies using a synthetic peptide reflecting amino acid positions 10-17 of the Hu-ras.sup.T24 gene product. The antibodies have been shown to react with the ras gene product p21, but were not shown to be specific for activated p21 (altered at position 12) rather than normal p21 protein.
Gallick, G. E. et al. on Sept. 4, 1983 published an abstract for a talk on the inhibition of transformation in a cell line infected with a temperature sensitive mutant of murine sarcoma virus by cytoplasmic microinjection of purified IgG from an antisera generated against a synthetic v-mos peptide. The transformation of the cells was induced by p85.sup.gag-mos fusion protein. The antibodies are not shown to be specific or an activated (mutant) oncogene product.
In Cancer Letter, Vol. 9 there is published a notice of an NIH RFP proposal for research on monoclonal antibodies to oncogene products of avian and mammalian retroviruses.
One difficulty in producing antibodies which are specific to single amino acid differences at position 12 between p21 proteins has been due to the complex structure of the protein, which did not allow antibodies to bind to the particular amino acid epitopic site desired. Another difficulty in producing such antibodies is the unpredictability of whether a given peptide segment will be effective as an immunogen in raising the necessary antibodies. Accordingly, there is a need in the art for an effective peptide immunogen to raise the appropriate antibodies and a method to obtain a modified, relaxed protein with an exposed epitope to which antibodies can bind so that single amino acid differences can be detected. Furthermore, there is a need to determine the mechanism of p21 protein activation so as to develop an effective therapeutic agent to counter the carcinogenic effects thereof.